fdhF is hypoxia-inducible promoter. In our project, it was used for initiation the expression of invasion gene and hyl gene. Under hypoxia environment it has a much higher efficiency of starting transcription than under normal 20% oxygen pressure. The starting efficiency of fdhF promoter is the key matter in our project. For detecting the starting efficiency of fdhF promoter, reporter gene GFP was connected to fdhF promote plasmid. And then the fluorescence intensity of GFP was detected under hypoxia environment.

1.1 Construction of fdhF induced GFP expression device

fdhF promoter (99bp) was synthesized by Genscript, nanjing, China. It was inserted into psB1C3 directly when synthesized. In order to detect the efficiency of this promoter at different oxygen concentration, we constructed the fdhF promoter induced GFP expression device. fdHF plasmid was digested by SpeI and PstI, while GFP geneator (BBa_E0840) was digested by XbaI and PstI. After purification the fragments, GFP was connected to the fdhF plasmid. The identification result was shown as below.

1.2 Sensitivity detection of fdhF promoter

After construction of fdhF induced GFP expression device, it was transformed into the E.coli. Under the hypoxia environment, we detected the fluorescence intensity of GFP. The O2 was limited to provide for the culture medium, so the pressure of O2 decreased down to the zero very quickly. Since the instrument can not detect the O2 below zero, we used the increasing of CO2 to stand for decreasing of O2. Some results are shown as below.

From the table 1, we can find that the pressure of O2 can not be detected when the E.coli were cultured after 4 hours. So we analyzed the corelation of GFP expression with the pressure of CO2. These data indicated that with the pressure of CO2 inscreasing, the starting efficiency of fdhF is increasing significantly, which means that fdhF promoter is very sensitive and efficient.

For detecting the sensitivity of the tetR repressible promoter, we built two expression vectors: the first one contains tetR repressor tagged with yBFP (J04500 + K642000 + B0015); another one is tetR repressible GFP expression device (R0040 + E0840) containing tetR repressible promoter and green fluorescent protain as a reporter.

2.1.Sensitivity of TetR for tetracycline concentration

For constructing of tetR repressor tagged with yBFP device, we connected three parts J04500, K642000 and B0015.

After construction, this plasmid was transferred into the competent cells (trans 5α). According to the BFP fluorescence would be affected when tetR combining with tetracycline, we added tetracycline with a series of concentration gradient to some of the tubes respectively with the same initial fluorescence intensity. Then we detected the changes of their fluorescence intensity of BFP at different time points. Consequently, we got the curve which shows the influence of the tetracycline concentration on the fluorescence intensity of tetR fusion protein. From the results, we can concluded that 20ug/ml tetracycline is the best concentration for inhibitiing of tetR expression.

2.2.Influence of tetracycline on the tetR inhibition to the tetR repressible promoter

For detecting the influence of tetracycline on the tetR inhibition to the tetR repressible promoter, we construct the tetR repressible GFP expression device (R0040 + E0840) which contains tetR repressible promoter and GFP as a reporter.

Fig.5 Identification of tetR repressible GFP device (R0040 + E00840).

After construction of these two plasmids, we transferred them into competent cells(trans 5α). According to their different resistances, we selected the clone containing these two plasmids. We added tetracycline with different concentration into LB medium to combine with tetR in order to remove the inhibition of tetR on the tetR repressible promoter, which indicates the more tetracycline was added, the stronger fluorescence intensity of GFP as well as the weaker fluorescence intensity of BFP. The sensitivity of tetR repressible promoter was detected by measuring the fluorescence intensity of GFP at different tetracycline concentrations and different time points.

The rsults showed that 20ug/ml tetracycline is the best concentration for inhibitiing of tetR expression in the double plasmids containing E.coli.

Fig.8 Monoclones on the agar plates shows that when only the tetR repressible GFP device was transformed into cells, GFP expressed very well. However, when both the tetR device and the tetR repressible GFP device are transformed into cell, GFP did not express GFP anymore, since it is inhibited by tetR.

TAT serves as an inducing factor in our project. TAT stands for "Trans-Activator of Transcription", serving as a trans-activating factor in HIV-1. TAT protein plays an important role in the HIV disease process. The protein is released by infected cells in culture, and is found in the blood of HIV-1 infected patients. It also can be absorbed by cells that are not infected with HIV, and act directly as a toxin producing cell death via apoptosis in uninfected. So, we constructed the TAT plasmid which can be expressed in Eukaryote to induce apoptosis of colon cancer in our project.

3.1 Construction and identification of TAT expression vector

TAT is synthesized by Genscript, Nanjing, China. The length of TAT is 309bp which was inserted into pUC57 plasmid. We amplied the TAT gene using PCR method, and the PCR product and pcDNA 3.1 were digested by the same double enzymes (EcoRI and PastI). After purification, TAT gene was connected with pCDNA3.1, forming TAT plasmid. Then we redigested the TAT plasmid for identification. The result is shown in Figure 9.

3.2 Function analysis of TAT plasmid in Eukaryote

For analyzing the function of TAT plasmid in Eukaryote, we extracted TAT plasmid from E.coli, and then transfected them into the colon cancer cell line LOVO, using Lipofectamine 3000. After transfection 48h, we used DAPI staining method to detect the apoptosis of cells. From the result we can see that the transfected cells showed apoptosis obviously. The results is shown in Figure 10.

Since our project aimed to use Nissle 1917 as the plasmid delivery, we also explored if Nissle 1917 is used for making yogurt in laboratory. We added the Nissle 1917 cells into the milk we bought in supermarket, stirred it evenly, and then put the milk into the incubator at the 37℃ overnight without shaking.

In the next morning, we found that the milk had been turned to semifluid which looks like yogurt. In order to further explore the changes occurred in the flask, we detected its pH value. This value is very similar with that of the yogurt bought from store, which indicating that it is possible to make yogurt using Nissle 1917. The result is shown as below.

Figure 11. The Nissle 1917 can make yogurt using milk after incubation overnight at 37 ℃. The left was not shaked in the next morning,while the right one was shaked. Figure 12. The pH value measurement for the yogurt made by Nissle 1917 (left) and the yogurt bought from supermarket (right), which shows two pH value are very similar.